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Reactive airways dysfunction syndrome is a form of irritant-induced asthma that has been documented after acute exposure to chlorine (Cl2) gas. Animal models of Cl2 exposure indicate that the airway epithelium is a target. gammadelta T-Cells are present in the mucosal surface of the airways and may control the growth and differentiation of the airway epithelial cells. However, the role of these cells in the airway response to Cl 2 exposure has not been elucidated. / Aim. To study the role of gammadelta T-cells in the response to Cl2 exposure with respect to inflammation and lung function. / Methods. C57B1/6J (wild type) and TCR-delta -/- mice exposed to Cl2 (400ppm) for 5 minutes were mechanically ventilated for measurement of responses to i.v. methacholine (MCh) at 1, 3, and 5 days after exposure. Bronchoalveolar lavage was performed to determine epithelial and leukocyte counts, and protein content. Tissues were harvested for PCNA immunoreactivity to evaluate the rate of repair of the epithelium. / Results. Wild type mice developed a greater degree of airway hyperresponsiveness to MCh at 1 day post exposure to Cl2 compared with TCR-delta-/- mice. Epithelial cell counts in BAL after Cl2 exposure were greater in TCR-delta -/- mice, and the pattern of inflammation differed in wild type and TCR-delta-/- mice; macrophages showed a later peak and granulocyte numbers were lower in TCR-delta-/- than in wild type mice. Both groups of mice had increased levels of total protein content in BAL after Cl2 exposure that resolved after 3 and 5 days, respectively. TCR-delta -/- mice have a lower rate of epithelial regeneration as shown by PCNA immunoreactivity. / Conclusion. The severity of airway injury after Cl 2 appears to be greater in TCR-delta-/- mice but the lack of TCR-delta seems to abrogate the changes in airway responsiveness to i.v. methacholine.

The N-terminal 236 residues of Ssa4p, a hsp70 in budding yeast, are sufficient to target GFP to nuclei in ethanol-treated cells; this transport is mediated by the karyopherin-beta Nmd5p. Ssa4p(1-236)-GFP nuclear accumulation upon ethanol exposure depends on the cell surface sensors Wsc1p and Mid2p as well as protein kinase C, an activator of the cell integrity MAPK cascade. I have analyzed the distribution of Ssa4p(1-236)-GFP and Nmd5p-His6-HA in deletion mutants of the cell integrity MAPK cascade and demonstrate that this pathway controls ethanol-induced nuclear accumulation of Ssa4p(1-236)-GFP. Protein phosphorylation regulates protein trafficking, and I have studied this modification for Ssa4p(1-236)-GFP, Nmd5p and the nucleoporin Nsp1p. My results suggest that the phosphorylation status of Nmd5p may change when cells are treated with ethanol, and threonine was identified as the putative target residue(s) for modification. Furthermore, Snf1p kinase is required for stress-induced nuclear accumulation of Ssa4p(1-236)-GFP; my data are in line with the idea that Snf1p kinase phosphorylates a threonine residue present at a consensus Snf1p site in Nmd5p. In summary, my studies link the stress-induced nuclear accumulation of Ssa4p(1-236)-GFP to the cell integrity MAPK pathway and Snf1p kinase.

The release of vasopressin (VP) from magnocellular neurosecretory cells (MNCs) of the supraoptic (SON) and paraventricular (PVN) nuclei is essential to hydromineral homeostasis. This release is controlled by several physiological stimuli, including changes in the osmotic pressure of the extracellular fluid, and in core body temperature. The osmotic control of VP release is mediated by specific and highly sensitive 'osmoreceptors'. Indeed, VP-releasing neurons in the SON are directly osmosensitive, and this osmosensitivity is mediated by stretch-inhibited cation channels. The molecular identity of these channels, however, remains unknown. The thermal control of VP release, on the other hand, is largely unexplained. In this thesis, we demonstrate that the mouse SON is a valid model for investigating the molecular basis of osmotransduction. We show that hyperosmotically-induced increases in membrane conductance are blocked by ruthenium red (RR), a non selective blocker of TRPV channels. In addition, SON neurons were found to express an N-terminal splice variant of TRPV1, but not full-length TRPV1. Unlike their wild-type counterparts, SON neurons in Trpv1 knockout (Trpv1-/-) mice could not generate RR-sensitive increases in membrane conductance and depolarizing potentials in response to hyperosmotic stimulation. Moreover, Trpv1-/-mice showed a pronounced serum hyperosmolality under basal conditions and severely compromised VP responses to osmotic stimulation in vivo. These results suggest that the Trpv1 gene may encode a central component of the osmoreceptor. Furthermore, we demonstrate that VP neurons are intrinsically thermosensitive. In these neurons, thermal stimuli spanning core body temperatures activate a RR-sensitive non selective cation current. Interestingly, VP neurons isolated from Trpv1 -/-mice are significantly less thermosensitive. These results suggest that channels encoded by the Trpv1 gene can confer thermosensitivity in the physiological range. Overall, these data suggest that products of the Trpv1 gene in VP neurons may represent a molecular point of convergence for the detection of osmotic and thermal stimuli.

We have identified receptor protein tyrosine kinases (PTKs) that are expressed and/or activated during kidney development. mRNA from fetal rat kidneys in late gestation (embryonic day 21), was used to prepare cDNA template for polymerase chain reaction amplification with primers based on conserved regions of PTKs, and products were subcloned and sequenced. Among 346 clones, we identified epidermal growth factor receptor (EGFr), Tie 2, platelet derived growth factor receptor (PDGFr)-alpha, PDGFr-beta, Flk-1, Flt-4, fibroblast growth factor receptor (FGFr)-1, FGFr-3, FGFr-4, Met, and RYK//Nbtk-1. PTK expression was studied by immunoprecipitation and immunoblotting of kidney membrane proteins with specific antibodies. EGFr, PDGFr-alpha, FGFr-1, FGFr-3, Met, and in some cases Tie-2 protein expression was greater in fetal kidneys, as compared with kidneys from 12 week adult rats, (controls). Flk-1, PDGFr-beta, and FGFr-4 proteins were expressed comparably; Flt-4 was not detected. As a reflection of receptor PTK activity, we assessed endogenous tyrosine phosphorylation, and in vitro autophosphorylation. EGFr and PDGFr-alpha displayed activity in fetal, but not adult kidneys. FGFr-3 and Flk-1 were active in some fetal kidneys; the other PTKs were not active. Thus, in late gestational rat kidney, there are distinct patterns of receptor PTK expression and activity. EGFr, PDGFr-alpha, FGFr-3 and Flk-1 are among PTKs that are activated, and they may mediate perinatal development of renal epithelial, interstitial, or vascular structures.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by eicosanoids. Recently, it has been demonstrated that the C5b-9 complex activates a high molecular mass cytosolic phospholipase A$ sb2$ (cPLA$ sb2)$ in cultured rat GEC, which leads to release of arachidonic acid (AA) and eicosanoids, and may contribute toward plasma membrane injury. In this study, we investigated if C5b-9 can also activate other PLA$ sb2$ isoforms. In GEC stably overexpressing sPLA$ sb2$ (Type II PLA$ sb2,$ 14 kD) (produced by transfection), the C5b-9-stimulated increase in free $ sp3$H-AA, ($ sim$2 fold above basal) was not significantly different from neo (control) GEC. In contrast, in GEC overexpressing cPLA$ sb2,$ C5b-9 stimulated a $ sim$9 -fold increase in free AA, as compared to unstimulated cells. This result suggests that C5b-9 activates cPLA$ sb2,$ but not the sPLA$ sb2$ enzyme, to release free AA. In further studies, we addressed the regulation of cPLA$ sb2$ in C5b-9-stimulated GEC. The present model of cPLA$ sb2$ activation involves the combined effects of: (1) an increase in cytosolic calcium concentration, which induces translocation of cPLA$ sb2$ from the cytosol to the plasma membrane, and may be mediated by the amino terminal "CaLB" (Ca$ sp{+2}$-dependent lipid binding) domain, and (2) phosphorylation by MAP kinase. To test the role of the CaLB domain, release of AA was monitored in GEC stably transfected with three constructs; cPLA$ sb2$ wild type (wt), cPLA$ sb2 Delta$CaLB (i.e., cPLA$ sb2$-wt with deleted CaLB domain), and neo (control) GEC. In GEC overexpressing cPLA$ sb2$-wt, C5b-9 stimulated a marked increase in free AA (as above). In contrast, in GEC overexpressing the cPLA$ sb2 Delta$CaLB enzyme, the C5b-9 induced increase in free $ sp3$H-AA was comparable to neo, despite expression of cPLA$ sb2 Delta$CaLB at levels similar to cPLA$ sb2$-wt. The results indicate tha

The multiple indicator dilution technique was employed to examine the in vivo kinetics of oxygen transport in the liver, known for its very permeable capillary bed, and the brain, known for its very tight capillary bed. $ rm sp{18}O sb2$-saturated $ sp{51}$Cr-labeled erythrocytes, labelled albumin, sucrose and water (the tracers for the study substance, vascular, interstitial, and cellular references respectively) were simultaneously injected into the target organs. Timed anaerobic samples were collected at the outflow and analyzed by mass spectrometry for relative $ rm sp{18}O sb2$ enrichment, and for $ gamma$ and $ beta$-radioactivity. Distributed-in-space models of capillary transport were used to analyze the data. / In the liver, oxygen transport and distribution were influenced by the temperature and hematocrit of the perfusate, and shown to be compatible with a flow-limited process. In the brain, flow varied significantly between animals, in concert with parallel changes in oxygen consumption. Analysis indicated that transfer of both $ rm sp{18}O sb2$ and $ sp3$H-water indicators from blood to brain is barrier limited, and it is concluded that low tissue oxygen concentration in the brain is due to limited endothelial permeability to oxygen.

From the onset of internal pipping (i.e. embryonal breathing of air cell gas) until hatching, the chorioallantoic membrane (CAM) and the lungs work simultaneously to serve the metabolic needs of the embryo. (1) The carbon dioxide and oxygen exchange rates (V̊O2 and V̊CO2) through the lungs and the CAM were separately, but simultaneously, measured during the last two days of incubation (day 20--21), while ventilation (V̊E) was calculated from the measurements of pressure oscillations in the air cell. When the embryo's total metabolic rate was increased, V̊E was linearly proportional to lung V̊O2 and V̊CO2 and not to the embryo's total metabolic rate. (2) Tracheal pressure and changes in lung volume were quantified through mechanical ventilation of the embryo. The curled up posture of the embryo, the eggshell and its membranes did not represent a significant mechanical constraint to V̊E. (3) V̊E, lung V̊O2 and V̊CO 2 were measured while the CAM compartment was exposed to either 10% O2, 100% O2 or 5% CO2. Total V̊O2 was also measured under these conditions. There is a clear V̊E-sensitivity to CO2 and a rather weak V̊E-sensitivity to changes in arterial oxygenation present at this stage of development.

The estrogen receptor (ER) is a ligand dependent transcriptional activator that belongs to the steroid/nuclear receptor superfamily. To probe the structure and function of the ER ligand binding domain (LBD), we developed a genetic screen in yeast Saccharomyces cerevisiae using a library of reverse ERs screened with a low affinity estrogen agonist, 2-methoxyestrone. Mutants isolated from this screen demonstrated altered ligand binding and/or transactivation properties. One of these mutants, L536P, showed high levels of constitutive activity in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. This suggests that substitution of a proline at position 536 in the wild type ER (HEGO) induces a reversible conformational change in the region of AF-2 that partially mimics activation of the receptor by hormone binding. / Activation of transcription by the ER requires the recruitment of different classes of coactivators. L536P interacted with coactivators in the absence of hormone and this constitutive interaction can be abolished by antiestrogens. We conclude that constitutive activity of L536P-HEGO is manifested to in part from constitutive coactivator binding. We also demonstrated that different classes of coactivators do not recognize the ER LBD in the same manner and can compete for binding to the ER LBD suggesting that different classes of coactivators recognize distinct but overlapping binding sites. Interestingly, coexpression of RIP140 blocked enhanced transactivation by HEGO observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. / Using a yeast two-hybrid system with the ER LBD as bait, we isolated a novel estrogen receptor cofactor (ERC) that interacts with the ER LBD in a hormone dependent manner. The primary structure of ERC consists of 4 ankyrin repeat domains, 2 LXXLL motifs and an ATP/GTP binding domain. ERC is highly expressed in ovary, testes, and spleen, with moderate levels in heart, brain, and placenta. ERC repressed ER transactivation in several cell lines in the presence of estradiol suggesting that ERC may function as a novel estrogen dependent repressor of the ER. Immunohistochemistry and confocal microscopy localized ERC to the cytoplasm with partial nuclear staining. Recently, synphilin-1, a novel protein that has been implicated in the pathogenesis of Parkinson's disease was isolated and is identical to ERC. A role for ERC in intracellular signaling through a membrane bound ER in the brain is currently being investigated.

This thesis examines the importance of the length-force relationship and the three-dimensional shape of the diaphragm with regard to its inspiratory function. As well as it reports on the manner in which fatigue and aminophylline affect the length-force properties of the diaphragm. First, I studied the effect of fatigue on diaphragm contractility as a function of sarcomere length using an in vitro rat diaphragm strips. Results indicated that fatigue resulted in disproportionately greater reduction of tetanic force at short sarcomere lengths. Second, I reconstructed the three-dimensional shape of the diaphragm to determine if in vitro results are physiologically relevant in humans. I estimated the changes in fibre length and shape that occurs with lung inflation from residual volume to total lung capacity in normal subjects. Results suggested that the inspiratory function of the human diaphragm can be entirely attributed to its length-force relationship rather than changes in shape under conditions of twitch phrenic nerve stimulations. Finally, I confirmed that fatigue caused a greater percent reduction of transdiaphragmatic pressure at high lung volume in response to single supramaximal shocks delivered bilaterally to the phrenic nerves at high lung volume; and demonstrated that aminophylline potentiated human diaphragm contractility more at high than at low lung volumes, both under fresh and fatigue conditions. I propose an explanation for the effect of fatigue and aminophylline on diaphragm contractility at different sarcomere lengths based on known actions of these factors and muscle shortening on excitation-contraction coupling mechanisms of skeletal muscles.